Studies with protein kinase C epsilon (PKC Epsilon) null mice indicate that PKC Epsilon regulates anxiety and alcohol consumption, dependence and reward. This appears to be related to actions at GABAA receptors since these mice are supersensitive to allosteric GABAA receptor agonists, including ethanol, in vivo and in vitro. The overall goal of this project is to understand how PKC Epsilon regulates GABAA receptor function. Studies will use immunohistochemistry and receptor binding autoradiography to compare the distribution of specific GABAA receptor subunits in wild type and PKC Epsilon null mice: Autoradiography will also be used to determine the affinity and density of binding sites for ligands that bind to benzodiazepine sites on specific receptor subpopulations. Western analysis will be used to measure the abundance of receptor subunits in limbic brain regions that express PKC Epsilon. L(tk-) fibroblast cell lines expressing different types of receptors will be examined to determine if PKC Epsilon regulation of allosteric sensitivity is specific for a particular subunit combination. Finally a PKC Epsilon mutant that can utilize novel ATP analogs as phosphate donors will be used to identify immediate substrates of PKC Epsilon in L(tk-) cells and in synaptoneurosomes, using immunoprecipitations with antibodies to GABAA receptor subunits and associated proteins, and 2D gel electrophoresis and mass spectrometry for unknown substrates. The hope is to define a PKC Epsilon pathway to further our understanding of GABAA receptor function as it relates to alcoholism.